Abstract Clostridium difficile is a major cause of diarrhea in health care settings, with one-half million cases of infection, annually. Clostridium difficile infection and disease (CDI) usually occurs after antibiotic treatment, which eliminates much of the intestinal commensal microbiota and provides C. difficile the opportunity to colonize the gut. If the colonizing C. difficile produces toxins, many patients develop CDI and suffer from symptoms ranging from diarrhea to life-threatening pseudomembranous colitis. The incidence of CDI is on the rise, in large part because of increased use of antibiotics. Further, CDI recurs in ~20% of treated patients, making this a serious and expensive disease, for which there is no cure and no effective approved vaccine. The goal of this proposal is to develop a protective vaccine to prevent CDI and also, its transmission in the health care environment. While a few C. difficile vaccines are in Phase II/III clinical trials, they target the toxins, but will not prevent colonization of C. difficile in the intestinal mucosa, nor its transmission. We think that the optimal protective vaccine would be one that generates a strong mucosal IgA antibody response against toxins, and also one that prevents colonization of C. difficile. Papillomaviruses are small DNA viruses in which the major capsid protein, L1, assembles with the minor capsid protein, L2 into virus-like particles (VLP). These VLPs infect mucosal surfaces, are not infectious but are strongly immunogenic, making them ideal vectors for mucosal vaccines. We have developed papilloma pseudoviruses (PsV) from capsid proteins L1 and L2, that contain plasmids expressing the receptor binding domain (RBD) of the C. difficile toxins A and B, and we have preliminary data showing that these vaccines can induce neutralizing mucosal IgA and protect mice from challenge by C. difficile. In Aim 1, we will develop PsV expressing RBD of toxins A, B (from different strains) and binary toxin. We will immunize them in mice and hamsters. We will identify an adjuvant that will increase the mucosal Ab response and generate long-term immunologic memory in mice and hamsters, the gold standard for CDI animal models. In Aim 2, we will generate PsV expressing surface molecules FliD or high molecular weight SLP, molecules that will affect colonization, and determine if these vaccines can induce antibodies that inhibit colonization of C. difficile. In Aim 3, we will vaccinate mice and hamsters with a mixture of all the PsV and challenge with toxogenic C. difficile spores from different strains to determine if CDI and colonization of C. difficile are prevented. If successful, the mucosal vaccine will generate a robust neutralizing mucosal IgA response against both toxins and the surface antigens FliD and SLP. Such a vaccine would make a major contribution to improving the health care of thousands of patients in health care settings, and save billions of dollars in health care costs.